Pluripotent (undifferentiated) stem cells possess the ability to become virtually any cell type in the human body and therefore, in principal, could be used to replace damaged tissues in organs that have traditionally been thought not to have a significant potential for functional self-repair such as heart muscle, spinal cord, brain tissue and kidney. However, to implement these therapies, one must have the ability to identify, isolate and proliferate populations of pluripotent stem cells. The first problem is that current methods for identifying pluripotent stem cells are insufficient. We have identified a new marker of pluripotency that discriminates between two different cell types within a population that current methods identify as being all "pluripotent". Secondly, it is difficult to culture embryonic stem cells (ESCs) without initiating differentiation. Growing ESCs under current state of the art protocols will yield only 50-75% undifferentiated colonies. This is in large part due to the fact that they are grown over a layer of "feeder" cells which secrete poorly understood factors, some of which promote the growth of undifferentiated stem cells and others that undoubtedly trigger differentiation. Factors secreted by neighboring cells influence how pluripotent stem cells differentiate. For example, pluripotent stem cells can be influenced to differentiate into a particular cell type by growing them over tissues of the desired cell type. What is needed is a cell-free system for growing ESCs in which only discrete, well-characterized agents are added to drive their growth. We have succeeded in growing ESCs in a cell-free system by adding a single agent that activates a newly identified growth factor receptor on the surface of ESCs - conditioned media from feeder cells was not added. The resultant population was 100% pluripotent and was signaled to differentiate by the withdrawal of our novel agent. This is a major step toward understanding the molecular drivers that maintain pluripotency as well as those that initiate differentiation and will enable stem cell therapies that are currently out of reach. Thus the isolation and propagation of pure pools of pluripotent stem cells will be critical for therapeutic applications of stem cell therapy for the repair of brain and spinal cord injuries, diabetes, cardiac failure, retinopathies and further will enable a whole range of therapeutic interventions that are currently not possible. PUBLIC HEALTH RELEVANCE: Pluripotent (undifferentiated) stem cells possess the ability to become virtually any cell type in the human body and therefore, in principal, could be used to replace damaged tissues in organs that have traditionally been thought not to have a significant potential for functional self-repair such as heart muscle, spinal cord, brain tissue and kidney. However, to implement these therapies, one must have the ability to identify, isolate and proliferate populations of pluripotent stem cells. We have succeeded in growing ESCs in a cell-free system by adding a single agent that activates a newly identified growth factor receptor on the surface of ESCs - conditioned media from feeder cells was not added. [unreadable] [unreadable] [unreadable]